Variability in Environmental Conditions Strongly Impacts Ostracod Assemblages of Lowland Springs in a Heavily Anthropized Area

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Article Variability in Environmental Conditions Strongly Impacts Ostracod Assemblages of Lowland Springs in a Heavily Anthropized Area

, , Giampaolo Rossetti * †, Valentina Pieri † ‡, Rossano Bolpagni , Daniele Nizzoli and Pierluigi Viaroli

Department of Chemistry, Life Science and Environmental Sustainability, University of Parma, 43124 Parma, Italy; [email protected] (V.P.); [email protected] (R.B.); [email protected] (D.N.); [email protected] (P.V.) * Correspondence: [email protected] These authors contributed equally to this work. † Present address: Iren Laboratori S.p.A., Piacenza, Italy. ‡ Received: 6 October 2020; Accepted: 17 November 2020; Published: 21 November 2020

Abstract: The Po river plain (Northern Italy) hosts artiﬁcial, lowland springs locally known as fontanili, which provide important ecosystem services in an area dominated by intensive agricultural activities. Here we present a study carried out in 50 springs. Each spring was visited once from October 2015 to January 2016. The sampled sites were selected to include springs studied in 2001 and 2004, to evaluate changes in water quality and ostracod assemblages that possibly occurred over a period of 10–15 years, and explore the relationships between ostracod community composition and water physical and chemical variables. Our results showed a decrease in the chemical water quality especially, in springs south of the Po river, evidenced by high nitrate levels. Most of the studied springs showed a relevant decrease in dissolved reactive silica, probably related to recent transformations of either agricultural practices or crop typology. Ostracods were mostly represented by common and tolerant species, and communities were characterized by low alpha diversity and high species turnover. Water temperature and mineralization level were the most inﬂuential variables in structuring the ostracod communities. We stress the need to implement conservation and restoration measures for these threatened ecosystems, to regain their role as ecosystem services providers.

Keywords: groundwater dependent ecosystems; Northern Italy; hydrochemistry; nutrient stoichiometry; non-marine ostracods; ecosystem services

1. Introduction A large number of semi-artificial, groundwater-dependent ecosystems (GDEs), locally known as “fontanili”, occur in the Po and Venetian plains (Northern Italy) along the alluvial fans and terrace deposits of watercourses in the transition zone from higher to lower plain. Across this so-called spring belt, due to changes in both terrain slope and sediment grain size [1–4], groundwater outflows through aquifers under a natural hydraulic gradient occur, which are usually further facilitated by driving perforated pipes and soil excavation. Most of the fontanili can be classified as rheo-limnocrenic springs [5]. The prevalent morphology consists of a spring area, known as “head”, which is relatively deep and where the water is almost still, and a drainage channel through which water flows downstream. In order to maintain suitable hydrological conditions, these systems require periodic management to remove the aquatic vegetation and the accumulation of organic sediment.

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Lowland springs are often described as stable habitats, with modest changes in hydrological, thermal, chemical, and biological characteristics, as they are fed by aquifers that guarantee relatively constant conditions at seasonal and interannual timescales [2,6]. The fontanili were exploited over centuries as micro-climate regulators in the lowland grassland of Lombardy. Here, until the late 1960s, in the cold season, stable meadows (locally called ”marcite”) were submerged in water from lowland springs at nearly 10–12 ◦C, a temperature much greater than the atmospheric one and sufficient to allow a slight warming and to avoid water freezing. Therefore, the productivity of such meadows was higher compared to traditional lawns. Lowland springs are components of interconnected systems of lentic waters, streams and channels, where nutrients can be removed by both aquatic vegetation and microbial processes [7,8]. They host relict (palaeo-) endemics, i.e., cold-stenotherm plants and animal taxa which underwent altitudinal displacements from mountain areas during the Würm glacial expansion, whose persistence is strictly linked to the conservation of these peculiar “ecological islands” surrounded by human-dominated landscapes [9–11]. Overall, lowland springs are refuge areas for species suffering strong declines in the study area, e.g., pike (Esox lucius)[12]. Despite their great potential value as ecosystem services providers, poor or inappropriate management practices, excessive water abstraction, land reclamation due to agricultural activities and the expansion of grey infrastructures, the spreading of alien species and the impacts of climate change on hydrology are threatening the conservation status of lowland springs [13–16]. Moreover, agriculture affects surface and groundwater quality due to the application of fertilizers that are largely in excess compared with crop uptake, resulting in a sensible increase in nutrient concentrations in most of these GDEs [17,18]. The hydrochemical and ecological features of the fontanili are relatively well studied [2,19–21], especially in the central area of the Po river plain [17,22–24]. In particular, two studies have investigated the relationships between water chemistry and ostracod assemblages [10,15]. Ostracods (class Ostracoda) are small bivalved crustaceans occurring in almost all aquatic ecosystems, mostly in benthic and periphytic habitats. According to Meisch et al. [25], there are presently 2330 subjective species of non-marine ostracods in 270 genera, all belonging to the order Pocodocopida. The Italian non-marine ostracod fauna consists of about 160 species, and it is currently the most diverse in Europe [26]. Accumulating evidence indicates that non-marine ostracods differently respond to environmental conditions, and may be used as bioindicators [27–35]. Their potential as environmental indicators has long been recognized by palaeolimnologists, who infer temporal changes in the local environment from changes in the assemblages of calcified ostracod valves [36–38]. The present research aimed at comparing the current hydrochemical status and the composition of the ostracod communities of 50 lowland springs of the Po river plain with those reported in previous studies carried out in the period 2001–2004 [10,15]. The magnitude of changes was statistically estimated using univariate methods, both between and within different sub-catchments to identify possible spatial patterns. Particular attention was paid to the trends of the stoichiometric ratios of the major dissolved nutrients. In addition, the distribution patterns and the compositional turnover of the ostracod communities over the considered time period were investigated, and the relationships between ostracod occurrence and environmental variables were evaluated by multivariate analysis. The results of this study can be used to assess the often-presumed stability of lowland springs, in terms of weak or delayed responses to anthropic disturbance in their hydrochemical and biological characteristics, and to provide insights into the potential future trajectories of these threatened ecosystems.

2. Materials and Methods

2.1. Study Area The study area encompassed 59 lowland springs belonging to nine sub-catchments of the Po river, located in ﬁve provinces: Piacenza and Parma in the Emilia-Romagna region (Apennine side of

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The study area encompassed 59 lowland springs belonging to nine sub-catchments of the Po theriver, Po plain, located south in five of provinces: the Po river), Piacenza and Lodi, and Parma Cremona in the and Emilia Milano-Romagna in the region Lombardy (Apennine region side (Alpine of sidethe of Po the plain, Po plain, southnorth of the ofPo the river), Poriver) and Lodi, (Table Cremona S1, Figure and1 ).Milano Each springin the Lombardy was visited region once (Alpine between Octoberside of 2015 the Po and plain, January north 2016. of the Samples Po river) were (Table collected S1, Figure from 1). each Each active spring spring, was visited 29 in Emilia-Romagna once between (identiﬁedOctober 2015with and the codes January “PC” 2016. and Samples “PR” followedwere collected by a number) from each and active 21 in spring, Lombardy 29 in (codeEmilia- “S” followedRomagna by (identified a number). with All the the codes sampled “PC” sites and were“PR” followed also included by a number) in the studies and 21 byin Lombardy Rossetti et (code al. [10 ] and“S” Pieri followed et al. [by15 a] onnumber). springs All in the Emilia-Romagna sampled sites were and also Lombardy, included respectivelyin the studies (same by Rossetti site codes et al. as [10] and Pieri et al. [15] on springs in Emilia-Romagna and Lombardy, respectively (same site codes above). In Rossetti et al. [10], sampling took place between May and August 2001, and each spring as above). In Rossetti et al. [10], sampling took place between May and August 2001, and each spring was sampled once; in Pieri et al. [15], each spring was sampled twice, in summer (late June–early was sampled once; in Pieri et al. [15], each spring was sampled twice, in summer (late June–early September) and autumn (November) 2004. September) and autumn (November) 2004.

LOMBARDY

EMILIA- ROMAGNA

FigureFigure 1. 1.Upper Upper left left panel: panel: mapmap ofof ItalyItaly inin whichwhich the Lombardy and and Emilia Emilia-Romagna-Romagna regions regions are are indicated,indicated, as as well well as as the the grey grey area area corresponding corresponding toto thethe main panel. Main Main panel: panel: location location of ofsampling sampling sitessites (black (black dots) dots) in in the the provinces provinces of of Parma Parma andand PiacenzaPiacenza (Emilia (Emilia-Romagna)-Romagna) and and Lodi, Lodi, Cremona Cremona and and MilanoMilano (Lombardy). (Lombardy).

2.2.2.2. Field Field and and Laboratory Laboratory Techniques Techniques WaterWater samples samples were were collected collected fromfrom eacheach sitesite in the head of of the the spring. spring. Water Water temperature temperature (T), (T), conductivityconductivity at at 25 25◦C °C (EC), (EC), pH pH and and dissolveddissolved oxygenoxygen (DO) were were measured measured using using a amultiparameter multiparameter probeprobe (Hanna, (Hanna, Mod. Mod. HI HI 9828). 9828). Water Water samples samples for for chemical chemical analysis analysis were were immediately immediately filtered filtered (0.45 (0.45µ m) μm) and stored at 4 °C until they were analyzed. Within 24 h, samples were analyzed for total and stored at 4 ◦C until they were analyzed. Within 24 h, samples were analyzed for total alkalinity (TA)alkalinity (Gran titration;(TA) (Gran [39 titration;]), ammonia [39]), [ 40ammonia], nitrite [40 [39], ],nitrite nitrate [39 after], nitrate reduction after reduction to nitrite to over nitrite cadmium over columnscadmium [39 ],columns dissolved [39] reactive, dissolved silica reactive (DRSi) silica [39] (DRSi) and soluble [39] and reactive soluble phosphorus reactive phosphorus (SRP) [41]. (SRP) [41]. Qualitative ostracod samples were gathered by a 250 µm handnet, pulling it close to the sediment and sweeping it through the vegetation. Living samples were transferred to the Water 2020, 12, 3276 4 of 20 laboratory, where ostracods were sorted under a binocular microscope and then fixed in 90% ethanol. Only specimens allowing unambiguous identification (i.e., adults and last juvenile stages) were considered; for Neglecandona gr. neglecta and Herpetocypris sp., the specific allocation remained uncertain because, respectively, only females and early stages were available. Both soft parts (dissected in glycerine and stored in sealed slides) and valves (stored dry in micropaleontological slides) were checked for the taxonomic identification, using González Mozo et al. [42] for Herpetocypris, Mazzini et al. [43] for Ilyocypris, and Meisch [44] for the remaining taxa.

2.3. Data Analysis Box-plots were used to illustrate the range of values of physico-chemical variables in different spring groups and periods. The ordination of abiotic and biotic data was carried out through principal component analysis (PCA) and canonical correspondence analysis (CCA). PCA was performed on the matrix of log-transformed (except for pH) physical and chemical water variables to assess their influence on the springs’ water quality. The concentration of dissolved inorganic nitrogen (DIN) was the sum of nitrate, nitrite, and ammonia. The same matrix and the ostracod absence/presence data were used to assess possible relationships between ostracod distribution and environmental data by CCA. The significance of canonical axes was assessed by Monte Carlo permutation tests (9999 permutations). Pearson correlation was used to evaluate the correlation degree between environmental variables, and Student’s t tests and Mann–Whitney U tests were used to assess possible differences between means and medians of the measured variables, respectively. Statistical analyses were performed using PAST ver. 4.03 [45].

3. Results

3.1. Physical and Chemical Variables

3.1.1. Water Characteristics of Springs Nine (PR34, PR40, S04, S10, S14, S16, S18, S19 and S20) out of the 59 sites sampled in 2001 and 2004 were found dry during the survey carried out in 2015–2016. T ranged between 9.1 and 14.1 in the Apennine side springs (the only exception was PR39 with 5.1 ◦C), and from 13.8 to 17.6 ◦C in the Alpine side springs. The EC values showed a strong variability, with a median signiﬁcantly higher 1 (Mann–Whitney U test, p < 0.01) in the Apennine springs (range from 450 to 2037 µS cm− , median 1 1 1 731 µS cm− ) than in Alpine ones (range from 211 to 896 µS cm− , 481 µS cm− ) (Figure2). Oxygen undersaturation was frequently found, close to hypoxia (<30%) in PC03 and PC32. Concurrently, distinct oxygen oversaturation (>160%) was observed in PR01 and PR39. The pH values were slightly to moderately alkaline, between 7.3 and 8.2 (Figure2). WaterWater2020 2020, 12, 12, 3276, x FOR PEER REVIEW 55 of of 20 20

Figure 2. Box-plots showing comparison between water temperature (T), conductivity (EC), dissolved oxygenFigure (DO) 2. Box and-plots pH showing values measured comparison in the between present water study temperature and in previous (T), conductivity research [10, 15(EC].) The, dissolved boxes show the 25th and 75th percentile (interquartile) ranges. Median values are shown as a horizontal black oxygen (DO) and pH values measured in the present study and in previous research [10,15]. The bar in each box. The whiskers extend up from the top of the box to the largest value that is 1.5 times boxes show the 25th and 75th percentile (interquartile) ranges. Median values are shown≤ as a the interquartile range, and down from the bottom of the box to the smallest value that is >1.5 times horizontal black bar in each box. The whiskers extend up from the top of the box to the largest value the interquartile range. Values outside this range are considered as outliers and are represented by that is ≤1.5 times the interquartile range, and down from the bottom of the box to the smallest value dots. SUM: summer; AUT: autumn. No DO data available for springs of Lombardy in 2004. that is >1.5 times the interquartile range. Values outside this range are considered as outliers and are represented by dots. SUM: summer; AUT: autumn. No DO data available for springs of Lombardy1 in The TA values were typical of well-buffered waters, with a minimum of 2.14 meq L− and a 2004. 1 maximum of 8.44 meq L− . On average, SRP concentrations were slightly lower in the Apennine side 1 1 springs (usually from below the detection limit to 20 µg P L− , except for isolated peaks up to 45 µg P L− ) −1 The TA values were typical of well-buffered waters, with a minimum 1of 2.14 meq L and a compared to the Alpine side springs, where values ranged between 3 and 42 µg L− . Nitrate represented maximum of 8.44 meq L−1. On average, SRP concentrations were slightly lower in the Apennine side on average >99% of DIN. Springs in the Apennine side had significantly higher (Mann–Whitney U test, −1 springs (usually from below the detection limit to 20 µg P L , except for1 isolated peaks up to 145 µg P p < 0.05) median DIN concentrations (range from 3.43 to 20.82 mg N L− , median 9.62 mg N L− ) than −1 −1 L ) compared to the Alpine side springs, where values ranged1 between 3 and 421 µg L . Nitrate springs in the Alpine side (range from 1.04 to 17.02 mg N L− , median 3.38 mg N L− ). Furthermore, represented on average >99% of DIN. Springs in the Apennine side had significantly higher (Mann– the DRSi concentrations had medians significantly larger (Mann–Whitney U test, p < 0.01) in the −1 Whitney U test, p < 0.05) median DIN concentrations1 (range from 13.43 to 20.82 mg N L , median 9.62 Apennine side (range between 1.18 mg Si L− and 5.93 mg Si L− ) than in the Alpine side (range −1 −1 −1 mg N L ) than springs1 in the Alpine side1 (range from 1.04 to 17.02 mg N L , median 3.38 mg N L ). between 0.51 mg Si L− and 2.71 mg Si L− ) (Figure3). Furthermore, the DRSi concentrations had medians significantly larger (Mann–Whitney U test, p < Water 2020, 12, x FOR PEER REVIEW 6 of 20

0.01) in the Apennine side (range between 1.18 mg Si L−1 and 5.93 mg Si L−1) than in the Alpine side (rangeWater 2020 between, 12, 3276 0.51 mg Si L−1 and 2.71 mg Si L−1) (Figure 3). 6 of 20

Figure 3. Box-plots showing comparison between TA, SRP, DIN, and DRSi values measured in the Figurepresent 3. study Box- andplots in showing previous comparison research [10 between,15]. Symbols TA, SRP, of box-plots DIN, and are DRSi as in values Figure 2measured. in the present study and in previous research [10,15]. Symbols of box-plots are as in Figure 2. 3.1.2. PCA Results 3.1.2. PCA Results The ﬁrst two axes of the PCA account for 74.70% of the total cumulative variance. PCA axis 1 was primarilyThe first deﬁned two byaxes loading of the of PCA DIN account (0.96) and for EC74.70% (0.82), of and thePCA totalaxis cumulative 2 by loading variance. of SRP PCA (0.73) axis and 1 wasT (0.35) primarily (Figure defined4). by loading of DIN (0.96) and EC (0.82), and PCA axis 2 by loading of SRP (0.73) and T (0.35) (Figure 4). Water 2020, 12, x FOR PEER REVIEW 7 of 20 Water 2020, 12, x FOR PEER REVIEW 7 of 20 Water 2020, 12, 3276 7 of 20

SRP PC03

S24 S06 S12 S17 PR04

%) DIN S27 S26

24 S25 . S09 S01 PR27

14 S15 PR31 PC46 ( S08 T S13 S02 2 S22 TA EC PR17 PC32 S05 pH S03 PC51 PC76 PR26 PR14 PR32 DRSi S07 PR33 PC47 PR10 S11 DO S28 PR09 PR29

Component Component PR01 PR13 PR06 PC75 PR39 S23

Component 1 (60.46%) Figure 4. Principal component analysis (PCA) diagram representing the ordination of springs in FigureFigure 4. 4. PrincipalPrincipal componentcomponent analysisanalysis (PCA) (PCA) diagram diagram representing representing the the ordination ordination of of springs springsrelation in in to environmental variables. T: temperature; EC: electric conductivity; TA: total alkalinity; DO: relationrelation to to environmental environmental variables. variables. T:T: temperature;temperature; EC: EC: electricelectric conductivity;conductivity; TA:TA: totaltotal alkalinity;alkalinity; DO:DO:dissolved oxygen saturation; SRP: soluble reactive phosphorus; DRSi: dissolved reactive silica; DIN: dissolveddissolved oxygenoxygen saturation;saturation; SRP:SRP: solublesoluble reactivereactive phosphorus;phosphorus; DRSi:DRSi: dissolveddissolved reactivereactive silica;silica; DIN:DIN:dissolved inorganic nitrogen. Point symbols refer to sub-catchments (Chiavenna: ; Arda-Ongina: +; dissolveddissolved inorganic inorganic nitrogen. nitrogen. PointPoint symbolssymbols referrefer toto sub-catchmentssub-catchments (Chiavenna:(Chiavenna: ; Arda-Ongina:; Arda-Ongina:Staffora-Luria-Versa-Coppa:+ +;; ; Taro: ; Parma: X; Enza: ; Lambro-Olona meridionale: ; Adda: • StaStafforaffora-Luria-Versa-Coppa:-Luria-Versa-Coppa: ; Taro:; Taro: ; Parma:; Parma: X; Enza:X; Enza:;  Lambro-Olona; Lambro-Olona meridionale: meridionale:; Adda:; Adda:;; Po: ). Po:; Po:). ). # 3 4 In the plane formed by the first two axes, springs located in the same sub-catchment generally InIn the the plane plane formed formed by by the the first first two two axes, axes, springs springs located located in thein the same same sub-catchment sub-catchment generallydo generallynot form do well-defined individual clusters. The springs of the Arda-Ongina, Staffora-Luria-Versa- notdo formnot form well-defined well-defined individual individual clusters. clusters. The springs The springs of the of Arda-Ongina, the Arda-Ongina, Staffora-Luria-Versa-Coppa Staffora-LuriaCoppa-Versa and Taro- sub-catchments, and most of those of the Enza, are located in the positive part of the andCoppa Taro and sub-catchments, Taro sub-catchments, and most and of most those of of those the Enza,of the areEnza, located are located in the in positive the positive partfirst of part theaxis, of first the due to the presence of more mineralized waters and, in particular, of higher DIN axis,first due axis, to due the presence to the presence of more mineralized of more mineralized waters and, waters in particular, and, in of higher particular, DIN concentrations.of concentrations. higher DIN The sites of the Adda sub-catchment are distributed in the third and fourth quadrants, Theconcentrations sites of the. The Adda sites sub-catchment of the Adda sub are-catchment distributed are in distributed the third andin the fourth third and quadrants, fourthdenoting quadrant denoting relativelys, low concentrations of DIN and a marked variability for SRP. Except for PR17 and relativelydenoting lowrelatively concentrations low concentrations of DIN and of DIN a marked and a marked variability variability for SRP. for Except SRP. forExcept PR17 forPR20, and PR17 PR20,the and springs of the Parma sub-catchment are located in the third quadrant, indicating lower thePR20, springs the springs of the Parma of the sub-catchment Parma sub-catchment are located are in located the third in quadrant, the third indicatingquadrant, lowerindicatingcontents contents lower of of both dissolved nitrogen and phosphorus compared to other springs in the study area bothcontents dissolved of both nitrogen dissolved and nitrogen phosphorus and compared phosphorus toother compared springs to inother the studysprings area in (Figurethe(Figure study4). 4).area (Figure 4). 3.1.3. Comparison with Previous Studies 3.1.3. Comparison with Previous Studies 3.1.3. Comparison with Previous Studies The springs of the study area had a relative thermal stability, although the T values notablyThe springs of the study area had a relative thermal stability, although the T values notably deviatedThe from springs the averageof the study in some area springs had a of relative the Apennine thermal side stability, (Figure 2although). Conversely, the T the values comparisondeviated notably from the average in some springs of the Apennine side (Figure 2). Conversely, the ofdeviated aggregated from chemical the average data with in someprevious springs studies of showed the Apennine different side levels (Figure of variability 2). Conversely, incomparison both sides the of aggregated chemical data with previous studies showed different levels of variability ofcomparison the watershed. of aggregated chemical data with previous studies showed different levels ofin variabilityboth sides of the watershed. in bothThe sides most of evident the watershed. changes in the Apennine side springs between 2001 and 2015–2016The can bemost evident changes in the Apennine side springs between 2001 and 2015–2016 can be observedThe formost EC evident and TA, changes DIN, and in DRSi the Apennine (Figures2, 3side and 5springs). A positive between linear 2001 correlation and 2015 between–observed2016 can TA forbe EC and TA, DIN, and DRSi (Figures 2, 3 and 5). A positive linear correlation between p andobserved EC was for found EC and in both TA, theDIN Apennine, and DRSi and (Figures Alpine sides2, 3 and of the 5). PoA positive river (r = linear0.49, correlation< 0.01 andTA andbetween r = 0.94,EC was found in both the Apennine and Alpine sides of the Po river (r = 0.49, p < 0.01 and r p TA< 0.01, and EC respectively). was found Anin both increase the Apenni in the meanne and values Alpine of side EC,s TAof the and Po DIN, river and (r = a 0.49, decrease p= < 0.94, 0.01 in DRSi, pand < 0.01, r respectively). An increase in the mean values of EC, TA and DIN, and a decrease in a=ff ected0.94, p all < 0.01, the sub-catchments, respectively). An although increase thesein the variations mean values were of notEC, alwaysTA and significant; DIN, and aDRSi, in decrease addition, affected in all the sub-catchments, although these variations were not always significant; in thereDRSi was, affected a strong all variability the sub-catchments, among sub-catchments although these as regards variations the were mean not values always of these significant;addition, variables. there in was a strong variability among sub-catchments as regards the mean values of these Theaddition, DRSi to there DIN was molar a strong ratio was variability similar or among slightly sub lower-catchments in 2015–2016 as regards compared the tomean 2001 values invariables. the springsof these The DRSi to DIN molar ratio was similar or slightly lower in 2015–2016 compared to 2001 ofvariables. the Chiavenna The DRSi sub-catchment to DIN molar and ratio in threewas similar springs or of slightly the Enza low sub-catchment.er in 2015–2016 All compared thein remaining the to springs 2001 of the Chiavenna sub-catchment and in three springs of the Enza sub-catchment. All springsin the springs underwent of the a markedChiavenna decrease sub-catchment in the ratio and over in thethree considered springs of period; the Enza the sub most-catchment. pronouncedthe remaining All springs underwent a marked decrease in the ratio over the considered period; the most changethe remaining occurred springs in PR39, underwent from 31.26 a tomarked 0.79, a decrease variation in that the was ratio however over the clearly considered anomalous period;pronounced compared the most change occurred in PR39, from 31.26 to 0.79, a variation that was however clearly topronounced those of the change other springs occurre ind the in investigated PR39, from area.31.26 to 0.79, a variation that was howeveranomalous clearly compared to those of the other springs in the investigated area. anomalous compared to those of the other springs in the investigated area. Water 20202020,, 1212,, 3276x FOR PEER REVIEW 8 of 20

Figure 5. Comparison between DIN and DRSi concentrations, and DRSi to DIN molar ratio (except for PR39,Figure which 5. Comparison scores lie outsidebetween the DIN graph) and forDRSi springs concentrations, of Emilia-Romagna and DRSi and to DIN Lombardy molar inratio 2015– (except 2016 andfor PR39, in previous which studies.scores lie Note outside that the axis graph) scales for are springs diﬀerent of for Emilia each- graph.Romagna and Lombardy in 2015– 2016 and in previous studies. Note that axis scales are different for each graph. In the springs of the Alpine side, EC, and to a lesser extent also DIN, showed substantial stabilityIn the between springs the of twothe Alpine study periods; side, EC on, and the to contrary, a lesser theextent other also variables DIN, show exhibiteded substantial evident changes,stability inbetween particular the DRSitwo study and SRP, periods; the latter on the often contrary, being belowthe other the detectionvariables limitexhibited of the evident analytical changes, method in (Figuresparticular2, 3DRSi and5 and). A SRP, decrease the latter in DRSi often to DINbeing molar below ratio the from detection 2004 andlimit 2015 of the was analytical observed method in all the(Figures springs 2, 3 (Figure and 5).5 ),A anddecrease the SRP in DRSi was stronglyto DIN mo imbalancedlar ratio from with 2004 respect and to 2015 DRSi was and observed DIN in bothin all periods.the springs A particularly (Figure 5), highand the variability SRP was was strongly found inimbalanced DIN and DRSi with concentrations. respect to DRSi The and large DIN standard in both deviationsperiods. A compared particularly to means high variabilityindicated that was most found hydrochemical in DIN and variables DRSi concentrations. exhibited high variability,The large evenstandard among deviatio springsns compared within close to geographicmeans indicated proximity that most in the hydrochemical same sub-catchment variables (Table exhibited1). high variability, even among springs within close geographic proximity in the same sub-catchment (Table 1). Water 2020, 12, 3276 9 of 20

Table 1. Comparison using Student’s t-test of means SD for selected physico-chemical variables in different sub-catchments of Emilia-Romagna (Apennine side) ± and Lombardy (Alpine side). Data from this study, [10] and [15]. For Lombardy, in 2004, only data collected in autumn are considered. N: number of springs; SL: significance level (NS: not significant; *: p < 0.05; **: p < 0.01); NA: not applicable.

EMILIA-ROMAGNA 1 1 1 1 EC (mS cm− ) TA (meq L− ) DIN (mg N L− ) DRSi (mg Si L− ) N 2001 This study SL 2001 This study SL 2001 This study SL 2001 This study SL Arda-Ongina 3 0.47 0.09 0.74 0.08 * 4.07 0.06 6.57 0.42 ** 7.33 3.27 16.12 6.01 NS 2.62 0.22 2.70 1.02 NS ± ± ± ± ± ± ± ± Chiavenna 5 0.42 0.04 0.70 0.13 ** 3.39 0.33 6.90 1.06 ** 5.89 1.62 8.00 8.59 NS 1.68 0.53 1.64 0.44 NS ± ± ± ± ± ± ± ± Staﬀora-Luria- 3 0.52 0.03 0.86 0.03 ** 4.93 0.33 7.52 0.34 ** 2.65 0.56 13.64 0.54 ** 5.29 2.31 2.38 0.78 NS Versa-Coppa ± ± ± ± ± ± ± ± Enza 6 0.43 0.11 0.70 0.04 ** 3.80 0.68 4.78 0.65 * 3.13 1.61 6.60 3.40 * 2.08 0.10 1.49 0.19 ** ± ± ± ± ± ± ± ± Parma 8 0.34 0.09 0.60 0.18 ** 3.03 0.67 4.57 1.14 ** 2.99 3.81 5.72 6.59 NS 6.19 2.31 2.54 0.78 ** ± ± ± ± ± ± ± ± Taro 4 0.98 0.63 1.27 0.58 NS 4.11 0.72 7.20 0.31 ** 5.89 1.67 13.58 4.29 * 10.98 1.71 3.68 1.66 ** ± ± ± ± ± ± ± ± LOMBARDY 1 1 1 1 EC (mS cm− ) TA (meq L− ) DIN (mg N L− ) DRSi (mg Si L− ) N 2004 This study SL 2004 This study SL 2004 This study SL 2004 This study SL Adda 15 0.39 0.04 0.45 0.05 ** 3.53 0.32 4.89 0.68 ** 3.33 0.93 2.94 1.02 NS 2.83 0.74 1.14 0.23 ** ± ± ± ± ± ± ± ± Lambro-Olona 4 0.43 0.14 0.52 0.21 NS 3.56 0.97 5.06 1.94 NS 5.22 2.63 4.58 2.44 NS 4.40 1.74 1.25 0.52 * meridionale ± ± ± ± ± ± ± ± Po 2 0.52 0.03 0.86 0.03 NA 4.51 0.08 6.91 0.77 NA 16.28 8.33 10.76 8.85 NA 7.73 2.09 2.10 0.85 NA ± ± ± ± ± ± ± ± Water 2020, 12, 3276 10 of 20

3.2. Ostracod Assemblages In total, 19 taxa, included in 12 genera in four families (Candonidae, Ilyocyprididae, Notodromadidae and Cyprididae), were identified (Table2). The mean number of taxa per site was 2.5 in Lombardy and 2.2 in Emilia-Romagna. The highest ostracod diversity (five taxa) was found in PR14, PR17 and S09; 14 springs hosted only one species, and sampling in PC32 did not yield any ostracod. The most frequent species were Cypria ophthalmica (in 37 sites), Cypridopsis vidua (14), and Prionocypris zenkeri (13). These three species, plus Ilyocypris inermis, I. bradyi and Herpetocypris reptans, were recorded in the whole study area. Candona candida, Herpetocypris brevicaudata, Heterocypris reptans, Ilyocypris gibba, Potamocypris smaragdina and Scottia pseudobrowniana were collected only in Lombardy. On the other hand, Cyclocypris laevis, Cyc. ovum, Candona gr. neglecta, Herpetocypris sp., Het. salina, Notodromas persica and Pseudocandona lobipes were exclusively found in Emilia-Romagna. Only seven sites, all located in Emilia-Romagna, did not show changes compared to previous surveys in their ostracod composition, possibly indicating a high compositional turnover in the communities of the studied springs. As for the diversity of the entire investigated area, out of a total of 25 identified ostracod taxa, 14 were reported both in this study and in the previous years, 5 only in this study, and 6 exclusively in earlier surveys; at a generic level, the sole difference was the occurrence of Potamocypris in this study (Table2). Water 2020, 12, 3276 11 of 20

Table 2. Comparison between ostracod assemblages found in 2015–2016 and in previous studies [10,15] in each sampling site. A: taxa recorded in all surveys; B: taxa absent in 2015–2016 but encountered in previous surveys; C: taxa found in 2015–2016 but absent in previous surveys. Σ: number of total records for each taxon in 2015–2016. sp. gr. sp. vra, 1891) á neglecta Site Code Sars, 1890 2015–2016 (Sars, 1887) Variation in (Koch, 1838) (Baird, 1835) Kempf, 1971 (Brady, 1868) (Brady, 1864) (V Gurney, 1921 (Jurine, 1820) (Jurine, 1820) (Hartwig, 1900) (Hartwig, 1901) Ilyocypris gibba (Ramdohr, 1808) Kaufmann, 1900 Kaufmann, 1900 Neglecandona Herpetocypris Ilyocypris bradyi Stepanaitys, 1960 Candona candida (Kaufmann, 1900) Cyclocypris ovum Ilyocypris inermis (O.F. Müller, 1776) Cyclocypris laevis Cypridopsis vidua Pseudocandona (O. F. Müller, 1776) (O. F. Müller, 1776) Heterocypris salina Notodromas persica Cypria ophthalmica Ilyocypris salebrosa Prionocypris zenkeri Heterocypris reptans Taxonomic Richness Taxonomic Richness (Chyzer & Toth, 1858) Herpetocypris reptans Neglecandona neglecta Pseudocandona lobipes Pseudocandona albicans Pseudocandona pratensis Scottia pseudobrowniana Potamocypris smaragdina Pseudocandona compressa Herpetocypris brevicaudata

PC03 BCA 2 = PC10 AA 2 = PC18 BA 1 –1 PC24 A 1 = PC32 BB 0 2 − PC44 AA C 3 +1 PC46 A A 2 = PC47 A C B 2 = PC51 A C B 2 = PC75 A 1 = PC76 AC 2 +1 PR01 A 1 = PR02 A 1 = PR03 A C C C 4 +3 PR04 A 1 = PR06 CA B C 3 +1 PR09 B C A 3 = PR10 B A B C C A 4 = PR13 CA A 3 +1 Water 2020, 12, 3276 12 of 20

Table 2. Cont. sp. gr. sp. vra, 1891) á neglecta Site Code Sars, 1890 2015–2016 (Sars, 1887) Variation in (Koch, 1838) (Baird, 1835) Kempf, 1971 (Brady, 1868) (Brady, 1864) (V Gurney, 1921 (Jurine, 1820) (Jurine, 1820) (Hartwig, 1900) (Hartwig, 1901) Ilyocypris gibba (Ramdohr, 1808) Kaufmann, 1900 Kaufmann, 1900 Neglecandona Herpetocypris Ilyocypris bradyi Stepanaitys, 1960 Candona candida (Kaufmann, 1900) Cyclocypris ovum Ilyocypris inermis (O.F. Müller, 1776) Cyclocypris laevis Cypridopsis vidua Pseudocandona (O. F. Müller, 1776) (O. F. Müller, 1776) Heterocypris salina Notodromas persica Cypria ophthalmica Ilyocypris salebrosa Prionocypris zenkeri Heterocypris reptans Taxonomic Richness Taxonomic Richness (Chyzer & Toth, 1858) Herpetocypris reptans Neglecandona neglecta Pseudocandona lobipes Pseudocandona albicans Pseudocandona pratensis Scottia pseudobrowniana Potamocypris smaragdina Pseudocandona compressa Herpetocypris brevicaudata

PR14 BCAAC A 5 +1 PR17 A B C A C C 5 +2 PR20 A B B C B 3 1 − PR26 A C A 3 +1 PR27 BA 1 1 − PR29 BAC 1 = PR31 CA 2 +1 PR32 BCA 2 = PR33 BCA 2 = PR39 A B B 1 -2 S01 CC 2 +2 S02 B A C B C B 3 1 − S03 ABBA 2 2 − S05 CAAB B 3 1 − S06 B B C C B C B 3 1 − S07 BBCB A 2 2 − S08 B B B C B B 1 4 − S09 B A C B C C B A 5 = S11 BC B B B 1 –3 S12 B B A A B A 3 3 − S13 BCAC 3 +1 S15 C C 2 +2 Water 2020, 12, 3276 13 of 20

S17 B B C B 1 2 − S21 BAAB A 3 = S22 AB B 1 –2 S23 AAA C 4 +1 S24 AACB C 4 +1 S25 BA A 2 1 − S26 ABA A 3 1 − S27 CBB B B C B C 3 2 − S28 BAB B 1 3 − Σ 1 4 5 37 14 5 7 1 1 1 7 2 4 0 0 3 5 1 11 0 0 2 0 0 1 Water 2020, 12, 3276 14 of 20

The relationships among abiotic variables and ostracod taxa are shown in the CCA triplot (Figure5). Because they were significantly correlated with EC (p > 0.05), four variables (TA, SRP, DIN and DRSi) were excluded from the analysis. The permutation tests indicated that the CCA was significant (Trace = 0.6918, p = 0.003), as well as the first three canonical axes (p < 0.05). The first two axes of the CCA ordination accounted for 51.51% and 30.78% of the total explained variance. Most of the taxa are grouped around the origin of the axes. The majority of the ostracods found only in the Emilia-Romagna springs are on the right side in the triplot, related to higher solute content. Heterocypris salina and Candona candida are clearly separated from the central cloud of points, and are in reciprocally opposite positions along both the first and second axis of the CCA. Heterocypris salina was recorded in a spring (PR03) with the highest conductivity value measured in the study area 1 (2037 µS cm− ), and Candona candida was recorded only in one site (S05), which was characterized by the lowest EC, TA and DRSi values in the entire study area.

4. Discussion The results of this study indicate a rapid worsening of environmental conditions in a number of lowland springs, as can be inferred from changes in water quality and, to a lesser extent, also in their hydrological and biological features. These aspects are discussed in detail, taking into account the context of growing anthropogenic pressures on agricultural soils and water resources, and the eﬀects resulting from their mutual interactions and feedbacks.

4.1. Effects of Hydrological Factors and Agricultural Practice on Water Quality This study, contrarily to what was previously described, highlights the marked variability of the environmental conditions of lowland springs, which are mainly linked to hydrological factors and multiple stressors operating at local scales and which, in turn, have repercussions for the composition and stability of the ostracod communities. In addition, the comparison with previous data shows that these ecosystems are subject to rapid changes, with a trend towards losses of functionality and reduced efficiency in negative feedbacks. Because of the more powerful aquifers that are recharged by water infiltration from Alpine watersheds compared to those in the Apennine area, the lowland springs in Lombardy have higher discharges and hydrological stability than the ones in Emilia-Romagna [46,47]. These latter, in fact, may undergo wide seasonal fluctuations in water level, and some may occasionally dry out due to water table lowering related to periods of low precipitation, intense water withdrawal for sprinkler irrigation and a prevalently intermittent hydrological regime of watercourses [48]. We therefore hypothesized that poor maintenance, and not water scarcity, is the main cause of drying in the seven springs in Lombardy, as reported in this survey. Water scarcity in the right side of the Po river basin may cause an increase in solute concentration when water demand is the highest and supply is the lowest, typically in summer, when typical local crops, like tomatoes, are grown. On the Alpine side of the Po basin, water is much more readily available thanks to the higher and more continuous flows of rivers, which are in some cases further guaranteed by water released from regulated subalpine lakes, as in the Adda river [48]. Therefore, in Lombardy, soil submersion and flood irrigation are very common. These techniques favor the recharge of the aquifers on which the springs depend, and thus also a gradual increase in their discharge rate. If soils are fertilized, irrigation also leads to a delivery of nutrients to groundwater; on the other hand, large irrigation water volumes can also increase solute dilution [49,50]. Different agricultural practices and crop types have varying effects on groundwater quality. In Emilia-Romagna, the autumn increase in DIN is compatible with nitrogen infiltration from soils fertilized in late summer for winter crop sowing (e.g., winter wheat and barley). Lower values in groundwater are likely due to either higher nitrogen uptakes by growing crops or possible denitrification. Higher DRSi values in summer can be related to a greater Si concentration due to the decrease in stored groundwater volumes, but also to infiltration from fields cultivated with cereals. Conversely, lower Water 2020, 12, 3276 15 of 20 values in autumn may depend on a higher dilution; the reduction in the quantities of manure which returned to fields and the lack of cereal straw burial can further exacerbate such a decrease in DRSi concentration [51]. In Lombardy, permanent meadows and corn (mostly harvested at a waxy maturation stage) prevail; DIN does not increase in autumn because there is no tillage for the sowing of winter cereals, and the low concentrations in DRSi may be due to dilution, the water that infiltrates into aquifers being low in Si [48–50].

4.2. Trends in Physico-Chemical Characteristics of Spring Waters The comparison of the physico-chemical water variables between both spring areas and surveys conducted in different years (Table1; Figures2 and3) must be considered with some caution due to the aforementioned differences in hydrological conditions, irrigation practices, crop types and, for Emilia-Romagna springs, site sampling periods. Nevertheless, some trends are detectable. Water temperature generally exhibited low variability, apart some evident deviations, both positive and negative, from average values in some springs of Emilia-Romagna (Figure2), where the groundwater is more influenced by meteoclimatic conditions due to small stored water volumes and/or reductions in aquifer recharge due to excessive withdrawal. A reduction in the outflows has the effect of transforming the springs from rheo-limnocrenic to more markedly lentic systems, with a consequent greater variability in the physical and chemical characteristics of the water. The positive linear correlation between TA and EC emphasizes how changes in solute concentrations are comparable in both the Apennine and Alpine sides of the Po river. They likely depend upon groundwater budgets, the water abstraction being responsible of water shortages and concentration increases, while the infiltration of soft water, e.g., from perialpine lakes in the Alpine side of the Po plain, may induce dilution. The other variables usually show marked changes (Table1). In particular, our results revealed a decline in water quality, more evident in the springs located in Emilia-Romagna, related to the “nitrogen legacy” from the past half century, when high nitrogen amounts were delivered to the farmland through massive applications of fertilizers and manure, resulting in a feedback loop between soil and water use [48,52,53]. In addition, in the whole study area a marked decrease in dissolved reactive silica is supposed to occur, related to changes in both cultivation and breeding practices and crop typologies in the last decades [51,54]. SRP concentrations were always very low, and often undetectable, because of an effective geochemical control of P availability due to the soils being rich in Ca and carbonates. In the Po river basin, N and P surpluses increased until the 1990s, then P markedly decreased, whilst N remained rather high [53]. The nutrient imbalance towards an N excess is further likely aggravated by the Si shortage, with potential impacts on aquatic vegetation/primary producers [51,54].

4.3. Structure, Distribution Patterns and Evolution of Ostracod Assemblages The ostracod communities of the studied lowland springs are generally composed of species that are commonly found in Northern Italy [26], and that are characterized by wide ecological tolerance. A notable exception is Ilyocypris salebrosa, which, however, was not found in the PR39 spring where it was present in 2001, and so far this is the only record of this species for Italy [26,43]. Some of the collected ostracod species (e.g., Herpetocypris brevicaudata, Ilyocypris bradyi, Ilyocypris inermis, Prionocypris zenkeri, Scottia pseudobrowniana) can be considered crenophilous, being usually associated to springs or waters connected to springs [44]. Scottia pseudobrowniana, recovered from mosses in S21, is restricted to semi-terrestrial conditions [44]. No stygobiont or non-native species were found in the sampled sites. Of the two species that are clearly separated from the central cluster of the CCA (Figure6), Heterocypris salina does indeed reﬂect its actual preference for salty waters, while the isolated position of the euryecious Candona candida simply depends on the physico-chemical characteristics of the only spring in which it occurs (S05). WaterWater 20202020,,12 12,, 3276x FOR PEER REVIEW 1615 ofof 2020

Can_ca DO Cyp_op Ily_gi Neg_ne Not_pe Pse_lo pH EC Ily_in Sco_ps 2 Cyp_vi

Pot_sm Axis Her_re Cyc_ov Pri_ze Cyc_la Her_br Ily_br Her_sp Het_sa Het_re T Axis 1

FigureFigure 6. Canonical correspondence ordination of ostracods and environmental variablesvariables onon the space defineddefined byby thethe firstfirst twotwo canonicalcanonical axes.axes. TheThe onlyonly significantsignificant variablesvariables ((pp< < 0.05)0.05) inin explainingexplaining speciesspecies occurrenceoccurrence are are displayed. displayed. Can_ca: Can_ca:Candona Candona candida candida; Cyc_la:; Cyc_la:Cyclocypris Cyclocypris laevis laevis; Cyc_ov:; Cyc_ov:Cyclocypris Cyclocypris ovum; Cyp_op:ovum; Cyp_op:Cypria ophthalmicaCypria ophthalmica; Cyp_vi:; Cyp_vi:Cypridopsis Cypridopsis vidua; Her_br: vidua; Her_br:Herpetocypris Herpetocypris brevicaudata brevicaudata; Her_re:; HerpetocyprisHer_re: Herpetocypris reptans; Her_sp: reptansHerpetocypris; Her_sp: Herpetocyprissp.; Het_re: Heterocypris sp.; Het_re: reptans Heterocypris; Het_sa: Heterocypris reptans; Het_sa: salina; Ily_br:HeterocyprisIlyocypris salina bradyi; Ily_br:; Ily_gi: IlyocyprisIlyocypris bradyi gibba; Ily_gi:; Ily_in: IlyocyprisIlyocypris gibba; inermisIly_in:; Ilyocypris Neg_ne: inermisNeglecandona; Neg_ne:gr. neglectaNeglecandona; Not_pe: gr. Notodromasneglecta; Not_pe: persica ;Notodromas Pot_sm: Potamocypris persica; Pot_sm: smaragdina Potamocypris; Pri_ze: Prionocypris smaragdina; zenkeri Pri_ze:; Pse_lo:PrionocyprisPseudocandona zenkeri; Pse_lo: lobipes ;Pseudocandona Pse_pr: Pseudocandona lobipes; Pse_pr:pratensis ;Pseudocandona Sco_ps: Scottia pratensis pseudobrowniana.; Sco_ps: Scottia pseudobrowniana. The ostracod assemblages of the lowland springs are generally simple, as they are expected to be inThe crenal ostracod habitats assemblages [10]. However, of the comparing lowland springs the specific are generally richness simple, with that as reportedthey are inexpected previous to studiesbe in crenal in the habitats same springs, [10]. However, it can be seencomparing that the the average specific number richness of specieswith that goes reported from 3.6 in in previous 2004 to 2.5studies in 2015 in the in Lombardy, same springs, and remainsit can be substantially seen that the stable average in Emilia-Romagna number of species (from goes 2.0 from in 2001 3.6 toin 2.2 2004 in 2015–2016);to 2.5 in 2015 in in both Lombardy, cases, these and are remains lower substantially values than those stable reported in Emilia for-Romagna the rheo-limnocrenic (from 2.0 in springs2001 to of the Palearctic region (4.7 2.7) [55]. The observed ostracod species diversity may be influenced 2.2 in 2015–2016); in both cases,± these are lower values than those reported for the rheo-limnocrenic bysprings the sampling of the Palearctic period. Pieri region et al. (4.7 [15 ] ± reported 2.7) [55] that. The out observed of the 16 ostracod ostracod species taxa collected diversity in lowland may be springsinfluenced from by Lombardy, the sampling 9 were period. present Pieri in et both al. [15 summer] reported and that autumn out of samples, the 16 ostracod 5 only in taxa summer collected and 2in only lowland in autumn. springs This from and, Lombardy, for some 9 species,were present the presence in both ofsummer larval stagesand autumn only, likely samples, indicates 5 only the in disummerfferent phenologicaland 2 only in behaviors autumn. This for ostracods and, for evensome in species, habitats the where presence water of temperature larval stages varies only, within likely aindicate narrows range.the different phenological behaviors for ostracods even in habitats where water temperature variesSome within diff aerences narrow inrange. the ostracod distribution exist between springs in Emilia-Romagna and Lombardy,Some possibly differences related in the to di ostracodfferent concentrations distribution exist of the between dissolved springs inorganic in Emilia fraction,-Romagna in particular and nitrogen,Lombardy, as evidenced possibly related by the results to different of the concentrationsCCA (Figure4). of Nevertheless, the dissolved no relationships inorganic fraction, between in ostracodparticular community nitrogen, as similarityevidenced andby the spatial results distance of the CCA were (Figure found. 4). TheNevertheless, low similarity no relationships in species compositionbetween ostracod between community geographically similarity proximate and spatial springs distance does not were seem found. to be The attributable low similarity to a low in dispersalspecies composition capacity of between ostracods. geographically Frequent disturbances proximate springs may lead does to not local seem extinctions to be attr followedibutable to by a colonizationslow dispersal fromcapacity nearby of ostracods. sites in spring Frequent ostracods disturbances communities, may lead according to local to extinctions source–sink followed dynamics; by undercolonizations such conditions, from nearby stochastic sites in processes spring ostracods might be morecommunities, influential according than environmental to source–sink characteristics dynamics; inunder shaping such ostracod conditions, assemblages stochastic during process earlyes recolonization might be more [56]. influential In addition, than over environmental a longer time scale,characteristics differences in shaping in environmental ostracod assemblages conditions occurringduring early in recolonization adjacent springs [56 may]. In addition, also determine over a thelonger occurrence time scale, of species differences which, in thoughenvironmental generally conditions widespread occurring in Northern in adjacent Italy, insprings our study may have also beendetermin showne the to occurrence belong to of a restrictedspecies which, number though of genera generally compared widespread to the in full Northern regional Italy, pool in [our26]. Thestudy above-stated have been shown hypotheses to belong are supported to a restricted by the number high speciesof genera turnover compared observed to the full in the regional study areapool (Table[26]. The2). above-stated hypotheses are supported by the high species turnover observed in the study area The(Table results 2). on ostracod assemblages only partially agree with those found for other fontanili-dwellingThe results on communities. ostracod assemblages Despite the only progressive partially worsening agree with of those their environmentalfound for other conditions, fontanili- thedwelling lowland communities. springs of the Despite Po river the plain progressive still act as worsening important refuge of their habitats environmental for both plant conditions, and animal the lowland springs of the Po river plain still act as important refuge habitats for both plant and animal Water 2020, 12, 3276 17 of 20 stenothermal and/or rare species [3,57–60]. However, swift changes in species and functionality were recently reported for fish communities in response to temperature increases, invasions of exotic fish, and habitat quality degradations [61].

5. Conclusions The lowland springs of the Po river plain can be regarded as important sentinels of environmental changes because the monitoring of their hydrological, hydrochemical and biological conditions provides useful information on the present status and trends of an area subjected to multiple interacting pressures. The alleged stability of the lowland springs is usually observed in the case of water temperature, while hydrochemical variables show more or less wide variations over a period of 10–15 years. Hydrological characteristics and agriculture practices cause clear differences in water quality and in ostracod composition, both between catchments and frequently also within the same sub-catchment, indicating a strong dependence of springs on local impacts. In general, the trends of the stoichiometric ratios of the major nutrients indicate breakdowns of the biogeochemical cycles due to multiple impacts. Ostracod communities are generally characterized by a low alpha diversity and a high species turnover, which seem to be indicative of severe disturbances due the variability of environmental conditions. The future climate is likely to further emphasize the fragility of these vulnerable ecosystems, as a result of a greater demand for water. Actions must be put in place to counter the deterioration or even the disappearance of these lowland springs, and more generally the GDEs, which constitute essential elements of the landscape of the Po valley, as well as aquatic biodiversity hotspots in a heavily modified agricultural area. An effective implementation of good agricultural practices to minimize the nitrate contamination, as well as contamination from the emerging contaminants of groundwater, is imperative. In addition, more effective guidelines for the correct maintenance of these ecosystems are needed to preserve or improve their hydraulic and ecological functionality. In this regard, a better integration between ecological and hydrogeological approaches in studying GDEs is essential so as to better understand the dynamics of these systems and their responses to climate change and human pressures.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4441/12/11/3276/s1, Table S1: Geographic characteristics of springs and sampling dates of the 2015-2016 survey. Author Contributions: All authors contributed equally to paper conceptualization, methodology setup and original draft preparation; V.P. and R.B.—sampling; D.N. and V.P.—sample analysis; G.R., V.P. and D.N.—data analysis and validation; G.R., D.N., R.B. and P.V.—review; G.R.—editing. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. V.P. was supported by the “Associazione Interregionale Partecipazione e Studi in Agribusiness Paesaggio e Ambiente” (IPSAPA) and the “Ecoistituto del Friuli Venezia Giulia” in the frame of the project “Studio della diversità dell’ostracodofauna delle acque dolci come indicatore di pressione antropica sui sistemi agro-alimentari” (2015–2016). Acknowledgments: Selena Ziccardi, Alessandro Scibona (University of Parma) are acknowledged for their support in different stages of this study. Conflicts of Interest: The authors declare no conflict of interest.

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